Western blot were performed as previously described [13 (link)]. The following primary antibodies have been used: anti‐actin (Cell Signaling, Danvers, MA, USA, 4970), anti‐Drp1 (BD Bioscience 611113), anti‐pS616‐Drp1 (Cell Signaling 4494), anti‐pS637‐Drp1 (Cell Signaling 6319), anti‐Mfn2 (Abcam ab56889), anti‐Mfn1 (Santa Crux sc‐50330), anti‐Opa‐1 (BD Bioscience 612607), anti‐Fis1 (Abcam ab71498), anti‐Mff (Abcam ab129075), anti‐pT202/204‐ERK1/2 (Cell Signaling 4370), anti‐ERK1/2 (Cell Signaling 4695), anti‐Hsp90 (Cell Signaling 4877), anti‐pSer2481‐mTOR (Cell Signaling 2974), anti‐mTOR (Cell Signaling 2983), anti‐MnSOD (Enzo Life Sciences ADI‐SOD‐110), anti‐LC3B (Cell Signaling 3868), anti‐cFos (Cell Signaling 4384), anti‐GAPDH (Cell Signaling 2118), anti‐Akt1 (Cell Signaling 2938), and anti‐pThr308‐Akt (Cell Signaling 4056). All primary antibody incubations were followed by incubation with appropriated secondary HRP‐conjugated antibodies (GE Healthcare or Cell Signaling) in 5% milk plus 0.1% Tween‐20 (Sigma P2287). Detection of protein signals was performed using Clarity Western ECL substrate (Bio‐Rad 170‐5061) and Amersham Imager 600. Stripping of the membranes for reprobing has been performed using buffer containing 1% Tween‐20 (Sigma P2287), 0.1% SDS (Sigma 71729), and 0.2 M glycine (VWR M103) at pH 2.2 (two washes for 10 min).
Anti akt1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-AKT1 is a primary antibody that recognizes AKT1 protein. AKT1 is a serine/threonine protein kinase that plays a key role in multiple cellular processes, including cell proliferation, survival, and metabolism.
Automatically generated - may contain errors
Lab products found in correlation
Anti akt2, by Cell Signaling Technology (7 mentions)
Anti akt3, by Cell Signaling Technology (6 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (5 mentions)
Anti erk1 2, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Beyotime (4 mentions)
Anti mtor, by Cell Signaling Technology (3 mentions)
Anti lc3b, by Cell Signaling Technology (3 mentions)
Anti gsk3β, by Cell Signaling Technology (3 mentions)
Anti foxo1, by Cell Signaling Technology (3 mentions)
Anti p21, by Cell Signaling Technology (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Anti gapdh, by Merck Group (3 mentions)
Anti h3, by Abcam (3 mentions)
Anti phospho akt1, by Cell Signaling Technology (3 mentions)
Anti phospho akt, by Cell Signaling Technology (3 mentions)
Anti p akt ser473, by Cell Signaling Technology (3 mentions)
Anti total akt, by Cell Signaling Technology (3 mentions)
Anti gapdh, by Santa Cruz Biotechnology (3 mentions)
Anti gapdh, by Cell Signaling Technology (2 mentions)
Amersham imager, by Cytiva (2 mentions)
41 protocols using anti akt1
1
Detailed Western Blot Methodology
2
Western Blot Analysis of Signaling Proteins
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Western blot was performed as previously described.3 (link) The anti-PARP, anti-p-β-catenin (S33/37/T41), anti-β-catenin, anti-c-Myc, anti-AKT1, anti-p-AKT1 (S473), anti-p-mTOR (S2448), anti-mTOR and anti-LC3B antibodies were purchased from Cell Signaling Technology (Beverly, MA). The anti-β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-SQSTM1/p62 antibody was from Abcam (Cambridge, UK). The densitometry of the immunoblots was performed with Image J software (NIH, Bethesda, MD)
3
Western Blotting and Immunofluorescence Analysis
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Western blots were carried out as described previously (Ventoso et al., 2006 (link)) using the following primary antibodies: anti-RPS4X (sc-85133, Santa Cruz Biotech.), anti-RPS7 (sc-377317, Santa Cruz Biotech.), anti-RPS6 (sc-74576, Santa Cruz Biotech.), anti-eIF4A (STJ2724, St. John´s lab,), anti-eIF3g (STJ23512, St. John´s lab), anti-EGFP (11814460001, Roche), anti-eEF1A1 (2551, Cell Signaling), anti-AKT1 (9272, Cell Signaling), anti-ACTB (T-5168, Sigma), anti-eIF4b (sc-376062, Santa Cruz Biotech.), anti-HRas (sc-53959, Santa Cruz Biotech.), anti-CCND3 (sc-453, Santa Cruz Biotech.), anti-ODC1 (sc-398116, Santa Cruz Biotech.), anti-GRK2 (a gift from C. Murga, CBMSO). Blots were developed with ECL (GE) and bands were quantified by densitometry. Immunofluorescence analysis was carried out as described previously using anti-RPS7 (1:500) and anti-mouse Alexa 595 as secondary antibody (Invitrogen). The preparations were analyzed under a Nikon A1R confocal microscope.
4
Western Blot Analysis of AKT/FoxO1/GSK3 Signaling
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Whole tissue protein extracts were prepared with NP-40 buffer (10 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP-40) containing protease inhibitors (SigmaFast, Sigma-Aldrich). Western blot analysis was carried out by standard methods[11 (link)]. Anti-phospho-AKT (Ser473) (#4058), anti-AKT1 (#2938), anti-phospho-FoxO1 (Ser256) (#9461), anti-FoxO1 (#2880), anti-phospho-GSK3-α/β (Ser21/9) (#9331), anti-GSK3-α (#9338), anti-GSK3-β (#9332) were purchased from Cell Signaling Technology (Danver, MA).
5
Western Blot Analysis of Protein Expression
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Protein separation and western blotting protocols were as described previously47 (link). Briefly, cells were lysed with RIPA solution, succeeded to SDS-PAGE gel electrophoresis, and the proteins transferred to PVDF membranes. The proteins were probed with anti-CISD2 (1:1,000 dilution; HPA015914, Sigma-Aldrich), anti-V5 (1:2,000 dilution; R960, Invitrogen), anti-GAPDH (1:10,000 dilution; MAB374, Millipore, Billerica, MA, USA), anti-E-cadherin (1:1,000 dilution; #3195, Cell Signaling Technology), anti-vimentin (1:1,000 dilution; #5741,Cell Signaling Technology), anti-ZO-1 (1:1,000 dilution; #5406, Cell Signaling Technology), anti-P21 (1:1,000 dilution; #2947,Cell Signaling Technology), anti-cytochrome C (1:1,000 dilution; GTX108585, GeneTex), anti-caspase 3 (1:1,000 dilution; GTX110543, GeneTex), anti-PARP1 (1:1,000 dilution; GTX100573, GeneTex), anti-EGR1 (1:1,000 dilution; #4153, Cell Signaling Technology), anti-PTEN (1:1,000 dilution; #9188, Cell Signaling Technology), anti-phospho-AKT Ser473 (1:1,000 dilution; (#4060, Cell Signaling Technology), and anti-AKT1 (1:1,000 dilution; #2938, Cell Signaling Technology) antibodies, and then further probed by horseradish peroxidase-conjugated secondary antibodies.
6
Protein Extraction and Western Blotting
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Cells were lysed for 30 min in Triton buffer (1% Triton X-100, 50 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS) supplemented with protease and phosphatase inhibitors (1 mM PMSF, 2 mM sodium pyrophosphate, 2 mM sodium betaglycerophosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 10 μg/ml leupeptin, and 10 μg/ml aprotinin). Lysates were cleared by centrifugation at 15,000 × g at 4°C for 15 min, and protein concentrations were determined via the Bradford method. 50 μg of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred onto Immobilon-P membranes. Proteins were detected by using anti-LETM2 (Proteintech, 17180-1-AP), anti-FGFR1 (Abcam cat# ab76464; RRID: AB_1523613 ), anti-WHSC1L1 (Abcam, ab180500), anti-CDCA7 (Abcam cat# ab69609; RRID: AB_1268064 ), anti-ERK1/2 (Santa Cruz, sc-514302), anti-p-ERK1/2 (Cell Signaling Technology cat# 4376; RRID: AB_331772), anti-AKT1 (Cell Signaling Technology cat# 2967; RRID: AB_331160), and anti-p-AKT1 (Cell Signaling Technology cat# 9018). Antibody binding was detected using horseradish peroxidase-labeled anti-mouse (Sigma) or anti-rabbit (Cell Signaling) antibodies and chemiluminescence was detected with a LAS4000 device (Fuji). Equal protein loading was confirmed with antibodies against GAPDH (Transgen).
7
Antibody-based Expression Analysis in Cancer Cells
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The following antibodies were used for immunofluorescence staining: anti-GAPDH (1:5,000; cat. no. Ab8245; Abcam); anti-UCP2 (1:100; cat. no. 11081-1-AP; ProteinTech Group, Inc.) and anti-FLNa (1:100; cat. no. ab76289; Abcam). The antibodies were used to detect UCP2 and FLNa expression in different CC cell lines.
The following antibodies were used for immunohistochemical staining: anti-UCP2 (1:200; cat. no. 11081-1-AP; ProteinTech Group, Inc.), anti-FLNa (1:200; cat. no. ab76289; Abcam), anti-P16 (1:200; cat. no. 10883-1-AP; ProteinTech Group, Inc.), and anti-Ki67 (1:1,000; cat. no. ab92742; Abcam).
The following primary antibodies were used for western blotting of relevant proteins: anti-UCP2 (1:1,000; cat. no. 11081-1-AP; ProteinTech Group, Inc.), anti-FLNa (1:1,000; cat. no. ab76289; Abcam), anti-GAPDH (1:5,000; cat. no. ab8245; Abcam), anti-ERK1/2 (1:1,000; cat. no. 4695; Cell Signaling Technology, Inc.), anti-P-ERK1/2 (1:1,000; cat. no. 4370; Cell Signaling Technology, Inc.), anti-AKT1 (1:1,000; cat. no. ab227100; Abcam), anti-P-AKT1 (1:10,000; cat. no. ab81283; Abcam), and anti-BCL-2 (1:1,000 cat. no. ab32124; Abcam).
The following antibodies were used for immunohistochemical staining: anti-UCP2 (1:200; cat. no. 11081-1-AP; ProteinTech Group, Inc.), anti-FLNa (1:200; cat. no. ab76289; Abcam), anti-P16 (1:200; cat. no. 10883-1-AP; ProteinTech Group, Inc.), and anti-Ki67 (1:1,000; cat. no. ab92742; Abcam).
The following primary antibodies were used for western blotting of relevant proteins: anti-UCP2 (1:1,000; cat. no. 11081-1-AP; ProteinTech Group, Inc.), anti-FLNa (1:1,000; cat. no. ab76289; Abcam), anti-GAPDH (1:5,000; cat. no. ab8245; Abcam), anti-ERK1/2 (1:1,000; cat. no. 4695; Cell Signaling Technology, Inc.), anti-P-ERK1/2 (1:1,000; cat. no. 4370; Cell Signaling Technology, Inc.), anti-AKT1 (1:1,000; cat. no. ab227100; Abcam), anti-P-AKT1 (1:10,000; cat. no. ab81283; Abcam), and anti-BCL-2 (1:1,000 cat. no. ab32124; Abcam).
8
Western Blot Antibody Validation
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For Western blot, the following antibodies were used: anti-KMT9α (#27630, lot 20062017, Schüle Lab, Freiburg, Germany, 1:500, rabbit), anti-KMT9β (#28358, lot 27022018, Schüle Lab, 1:500, rabbit), anti-PTEN (CST#9552, lot 3 08/2019, Cell Signaling, 1:500, rabbit), anti-TRP53 (#LIN-P956, lot 6065476, Linaris BIOZOL, Eching, Germany, 1:500, rabbit), anti-β-Actin (#A1978, lot 0000086304, Sigma, 1:10,000, mouse), anti-EGFR (#2232, lot 17, Cell Signaling, 1:1000, rabbit), anti-AKT1 (#2938, lot 4, Cell Signaling, 1:1000, rabbit), anti-GAPDH (#M20028M, lot 244888, Abmart, Berkeley Heights, NJ, USA, 1:5000, mouse), and anti-tubulin (alpha tubulin, #T6074, lot 024M4837, Merck KGaA (Darmstadt, Germany)., 1:10,000, mouse). Western blots were developed and quantified using an Amersham Imager 600 (GE Healthcare, Buckinghamshire, UK).
9
Histone Modifications in Brain Regions
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Frozen tissue punches of the relevant regions were homogenized and boiled in 1% SDS. For clozapine and quinpirole treatments tissues were obtained 24h after the last injection. Extracts (50 μg) were separated on SDS-PAGE and transferred onto membranes. Antibody used were: anti-H3K9me2 (1:5000), anti-H3K9me3 (1:1000) from Active Motif; anti-H3K4me3 (1:1000), anti-H3 (1:1000) from Abcam; anti-GAPDH (1:20,000) Millipore; anti-Akt1 (1:1000) Cell Signaling; anti-NR4A2 (1:1000) Antibody Verify. Secondary anti-rabbit and/or anti-mouse antibodies (1:5000) were from Millipore. Western blots were revealed using ECL Plus (GE Healthcare or Westdura, Pierce). Quantifications were performed using the NIH ImageJ (version 1.42q) software.
10
Immunofluorescent Analysis of Angiogenic Receptors
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Antigen retrieval was done in the same manner as IHC described above. After pretreatment, sections were blocked with 5% normal goat serum in T‐PBS for 1hr at room temperature and then incubated overnight at 4 °C with the following primary antibodies diluted in T-PBS: anti-Ephrin-B2 (Santa Cruz, SC1010 or SC19227), anti-Eph-B4 (R&D, AF446 or Santa Cruz, SC5536), anti-vWF (Abcam, ab1713), anti-alpha-actin (Abcam, ab5694), anti-phospho-tyrosine (Abcam, ab10321), anti-phospho-Akt1 (Cell Signaling, #9018), or anti-Akt1 (Cell Signaling, #2967). Sections were then treated with secondary antibodies at room temperature for 1–2 hr using goat anti-rabbit Alexa Fluor 488 (Life Technologies, Grand Island, NY), donkey anti-goat Alexa-Fluor-488 (Life Technologies), or donkey anti-rabbit Alexa-Fluor-568 (Life Technologies). Sections were stained with SlowFade® Gold Antifade Mountant with DAPI (Life Technologies) and coverslip applied. Digital fluorescence images were captured and intensity of immunoreactive signal was measured using Image J software (NIH, Bethesda, Maryland). Intensity of merge signal was determined by applying a color threshold selective for yellow signal.
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